Therapeutic potential of MEK inhibition in acute myelogenous leukemia: rationale for "vertical" and "lateral" combination strategies

: In hematological malignancies, constitutive activation of the RAF/MEK/ERK pathway is frequently observed, conveys a poor prognosis, and constitutes a promising target for therapeutic intervention. Here, we investigated the molecular and functional effects of pharmacological MEK inhibition in cell line models of acute myeloid leukemia (AML) and freshly isolated primary AML samples. The small-molecule, ATP-non-competitive, MEK inhibitor PD0325901 markedly inhibited ERK phosphorylation and growth of several AML cell lines and approximately 70 % of primary AML samples. Growth inhibition was due to G(1)-phase arrest and induction of apoptosis. Transformation by constitutively active upstream pathway elements (HRAS, RAF-1, and MEK) rendered FDC-P1 cells exquisitely prone to PD0325901-induced apoptosis. Gene and protein expression profiling revealed a selective effect of PD0325901 on ERK phosphorylation and compensatory upregulation of the RAF/MEK and AKT/p70( S6K ) kinase modules, potentially mediating resistance to drug-induced growth inhibition. Consequently, in appropriate cellular contexts, both "vertical" (i.e., inhibition of RAF and MEK along the MAPK pathway) and "lateral" (i.e., simultaneous inhibition of the MEK/ERK and mTOR pathways) combination strategies may result in synergistic anti-leukemic effects. Overall, MEK inhibition exerts potent growth inhibitory and proapoptotic activity in preclinical models of AML, particularly in combination with other pathway inhibitors. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective targeted strategies for the treatment of AML

A Relapsing Meningeal Acute Myeloid Leukaemia FLT3-ITD+ Responding to Gilteritinib

A patient with a therapy-related acute myeloid leukaemia (AML), NPM1(mut), and FLT3-ITD+ was treated with induction and consolidation with CPX-351, obtaining a complete response (CR) but minimal residual disease persisted positive. Later, she complained progressive burning leg pain, weakening of the right hand and leg muscles, associated with absence of osteotendinous leg reflexes. Examination of cerebrospinal fluid (CSF) showed a meningeal relapse of AML. Moreover, a magnetic resonance imaging (MRI) showed 2 right meningeal implants of myeloid sarcoma and bone marrow revealed haematologic relapse of disease. She was treated with medicated lumbar punctures (LPs) followed by an FLA-Ida scheme, and she achieved a 2nd CR. Unfortunately, the patient developed hyperleucocytosis and reappearance of meningeal myeloid sarcoma at MRI. For this reason, a monotherapy with gilteritinib (an FLT3 inhibitor) was started: after 3 months of therapy, central nervous system (CNS)-disease shrunken and then faded, while AML in the bone marrow achieved only a partial response. This is the 1st report of a positive biological effect of gilteritinib on CNS (meningeal) myeloid sarcoma. There are no studies of gilteritinib concentration into CSF and penetration of gilteritinib into the blood-brain barrier should be further studied, given the paucity of drugs active on CNS relapse of AML. In patients receiving CPX-351 only, diagnostic LP should be considered after induction

Cell processing for haplo-identical hematopoietic stem cell transplantation: Automated washing and immunomagnetic-positive selection

BACKGROUND AIMS: Immunomagnetic cell selection (ICS) of CD34(+) cells is being used increasingly in allogeneic transplantation in order to reduce T-cell quantity. The aim of this study was to evaluate an automated washing protocol before immunomagnetic selection. METHODS: The automated method was compared with a conventional washing procedure. In the study group the cell processing using the automated procedure, both before and after antibody incubation, was performed with a Sepax S-100 device. The efficacy of the automated procedure was compared with the control group, where washing were performed using a standard method. RESULTS: The results obtained after pre-incubation washing performed using the automated system showed a total nucleated cell (NC) and CD34(+) cell recovery of 84.87% (71.80-105, SD 8.62; range, standard deviation) and 83.45% (47-109, SD 16.12), respectively. The NC and CD34(+) cell recovery after the pre-incubation washing cycle was performed using the standard method was 75.54% (38.36-97.76, SD 22.5) and 61.51% (30.87-81.79, SD 19.3), respectively. The CD34(+) cell recovery after ICS was 51.27% (13.77-98.82, SD 24.97) and 48.89% (15.57-88.24, SD 25.91) for group 1 and group 2, respectively. The average purity in group 1 was 86.46% (67.4-96.10, SD 13.07) and in group 2 84.97% (58.1-97.8, SD 15.58). CONCLUSIONS: The efficacy of the ICS led to an optimal purity without affecting cell recovery, which was higher in group 1. Overall, our data suggest that the automated method is suitable for washing hematopoietic progenitor cell apheresis (HPC-A) concentrates before immunomagnetic cell selection in daily clinical routines

Automated washing of human progenitor cells: Evaluation of apoptosis and cell necrosis

BACKGROUND: High-dose chemotherapy followed by reinfusion of autologous stem cells harvested from peripheral blood has been increasingly applied for a variety of disorders. The critical importance of cell dose in the clinical outcome, after transplant, has motivated the need to develop techniques aimed at reducing cell losses and increasing reproducibility. OBJECTIVES: The aim of this study is to evaluate the efficacy of the Sepax S-100 device to process thawed HPC-A products in comparison with manual procedure. METHODS/MATERIALS: We have analysed viability, total nucleated cells (TNC), haematopoietic progenitors and CD34+ cells recovery. RESULTS: The TNC and CD34+ cells recovery in the automatic procedure was of 91.9% (73-100; SD ± 12.60) and 86.7% (69-100; SD ± 10.21), respectively. Instead the recovery of TNC and CD34+ cells using the manual method was of 84.7% (47-100; SD ± 22.9) and 80.29% (23-100; SD ± 25.96). The results, obtained from the assessment of viability of CD34+ both 7-AAD)+ and AnnV+ showed a high percentage of necrosis and apoptosis in this cell subset by using the manual procedure in respect to the Sepax automated system. CONCLUSION: Overall, our data suggest that the automated washing procedure is safe and suitable for processing of thawed HPC-A products and can be daily used in clinical routine

Systemic, Mucosal Immune Activation and Psycho-Sexual Health in ART-Suppressed Women Living with HIV: Evaluating Biomarkers and Environmental Stimuli

Gender medicine is now an approach that can no longer be neglected and must be considered in scientific research. We investigated the systemic and mucosal immune response in a population of women living with HIV (WLWH) who were receiving successful ART and the sexual and psychological repercussions of HIV infection on the women’s health. As control group, healthy women (HW) matched for age and sex distribution, without any therapy, were included. In summary, our study highlighted the persistence of immune-inflammatory activation in our population, despite virological suppression and a normal CD4 cell count. We found a hyperactivation of the systemic monocyte and an increase in inflammatory cytokine concentrations at the systemic level. The analysis carried out showed a significantly higher risk of HPV coinfection in WLWH compared to HW. Furthermore, our data revealed that WLWH have a profile compatible with sexual dysfunction and generalized anxiety disorders. Our study underlines that patients living with HIV should be evaluated by multidisciplinary teams. These findings also support the idea that more and different immunological markers, in addition to those already used in clinical practice, are needed. Further studies should be carried out to clarify which of these could represent future therapy targets

Therapeutic potential of MEK inhibition in acute myelogenous leukemia: rationale for "vertical" and "lateral" combination strategies.

In hematological malignancies, constitutive activation of the RAF/MEK/ERK pathway is frequently observed, conveys a poor prognosis, and constitutes a promising target for therapeutic intervention. Here, we investigated the molecular and functional effects of pharmacological MEK inhibition in cell line models of acute myeloid leukemia (AML) and freshly isolated primary AML samples. The small-molecule, ATP-non-competitive, MEK inhibitor PD0325901 markedly inhibited ERK phosphorylation and growth of several AML cell lines and approximately 70 % of primary AML samples. Growth inhibition was due to G(1)-phase arrest and induction of apoptosis. Transformation by constitutively active upstream pathway elements (HRAS, RAF-1, and MEK) rendered FDC-P1 cells exquisitely prone to PD0325901-induced apoptosis. Gene and protein expression profiling revealed a selective effect of PD0325901 on ERK phosphorylation and compensatory upregulation of the RAF/MEK and AKT/p70( S6K ) kinase modules, potentially mediating resistance to drug-induced growth inhibition. Consequently, in appropriate cellular contexts, both "vertical" (i.e., inhibition of RAF and MEK along the MAPK pathway) and "lateral" (i.e., simultaneous inhibition of the MEK/ERK and mTOR pathways) combination strategies may result in synergistic anti-leukemic effects. Overall, MEK inhibition exerts potent growth inhibitory and proapoptotic activity in preclinical models of AML, particularly in combination with other pathway inhibitors. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective targeted strategies for the treatment of AML

Consensus for Flow Cytometry Clinical Report on Multiple Myeloma: A Multicenter Harmonization Process Merging Laboratory Experience and Clinical Needs

Flow cytometry is a highly sensitive and specific approach for discriminating between normal and clonal plasma cells in multiple myeloma. Uniform response criteria after treatment have been established by the International Myeloma Working Group and the EuroFlow Group; however, the way in which flow cytometry data are reported has suffered from no collaborative or multicentre efforts. This study, involving 8 expert laboratories and 12 clinical hematology units of the Lazio region in Italy, aims to produce a uniform and shared report among the various Centres. From the pre-analytical phase to sample processing, data acquisition, analysis, and evaluation of the potential limitations and pitfalls of the entire process, the study reaches a final conclusion shared by laboratories and clinicians according to the most updated principles and recommendations. The aim was to identify the necessary data to be included in the clinical report by using multiple-choice questionnaires at every single stage of the process. An agreement of more than 75% of the laboratories was considered mandatory for the data to be included in the report. By ensuring the operational autonomy of each laboratory, this study provides a clear report that limits subjective interpretations and highlights possible bias in the process, better supporting clinical decision-making